Almost 20% of the population world-wide are suffering from allergy. Consequently, it is a health problem of increasing seriousness. Allergy is a hypersensitivity reaction against substances in air, food or water, which are normally harmless (Corry and Kheradmand, 1999). A new and foreign external agent triggers an allergic reaction, which aims at disposal of that agent from the body. In IgE-mediated allergic reactions, also called immediate or type I hypersensitivity reactions, under the first exposure of a foreign substance, allergen, to the body, IgE-bearing B-cells begin to produce soluble IgE molecules which will then bind to high-affinity IgE receptors present on the surface of a wide variety of cells, most importantly to mast cells and basophils. If the same foreign substance is encountered again, the cross-linking of the receptor-bound IgE molecules by the allergen occurs, resulting in cellular activation followed by the release of toxic products such as histamine, which will elicit the signs and symptoms of an allergic reaction.
Cow's milk allergy (CMA) is a most common cause of clinically important adverse food reactions with infants and children during the first 2 years of life (Savilahti, 1981; Host and Halken, 1990; Saarinen et al., 1999). It is characterized by a strong IgE response to milk proteins and clinical symptoms in skin and gastrointestinal tract such as atopic eczema, vomiting and diarrhoea (Vaarala et al., 1995; Saarinen, 2000). Symptoms in respiratory ducts and anaphylactic shock are also possible (Host and Halken, 1990; Schrander et al., 1993; Hill et al., 1999; Vanto et al., 1999; Saarinen, 2000). CMA is a serious problem with children, because milk is an important source of energy (up to 50%) for young children and is not very easily replaceable with non-dairy products. Nearly 85% of the milk allergic children will outgrow of their allergy by the age of 3, but remission of CMA may occur in up to one-third of older children (Sampson and Scanlon, 1989)
One of the major allergens in cow's milk is β-lactoglobulin, which belongs to the protein family known as lipocalins. Lipocalins consists a group of a small ligand binding proteins, mostly respiratory allergens such as Mus m1, Rat n1 (mouse and rat urinary proteins) and a German cockroach allergen Bla g4 (Rouvinen et al. 2001). β-lactoglobulin occurs naturally in the form of a 36 kD dimer with each subunit corresponding 162 amino acids. Totally six genetic variants of the β-lactoglobulin has been identified based on the sequence differences. The most prevalent variants A and B differ only at the position 64 (Asp→Gly) and 118 (Val→Ala) (Godovac-Zimmermann and Braunitzer, 1987). The 3D-structure of the β-lactoglobulin has been determined by X-ray diffraction (Sawyer L. et al, 1985, Brownlow, S. et al, 1997)
IgE antibodies distinctively recognise allergenic epitopes, which would be useful in clinics and immunodiagnostics for detecting and determining allergen concentrations of complex materials. Further, according to this invention, allergenic epitopes are usually different from the immunogenic epitopes of proteins. This fact has hampered the production of monoclonal antibodies capable of specific binding of allergenic epitopes by conventional methodology such as hybridoma technology. It has been recently shown that the development of allergen-specific IgE antibodies is possible by the phage display technology (Steinberger et al., 1996). This methodology is giving new tools to produce allergen-specific recombinant antibodies that can be produced in consistent quality for clinical and diagnostic applications.
The technical problem to which the present invention is related is the detection of actual binding sites of IgE antibodies in allergenic polypeptides and use of this information, e.g., to modify these polypeptides to decrease their allergenicity. Previous solutions for this problem are disclosed in U.S. Patent Application No. 2003/0175312 (Holm et al.), WO 03/096869 (Alk Abello A/S) and Jenkins et al. 2005 (J. Allergy Clin. Immunol. 115:163-170). In these documents, it is described that the putative IgE binding sites in allergenic polypeptides may be detected by sequence analysis of conserved surface structures of allergenic polypeptides. Further, in US 2005/0181446 (Roggen et al.) and Hantusch et al. 2004 (J. Allergy Clin. Immunol.) a peptide-scan approach is used to find IgE binding epitopes. However, none of these documents discloses the method of the present invention wherein an IgE binding site on an allergenic polypeptide is found based on the experimental 3D and molecular modelling data of a novel type of IgE epitope having essentially planar or flat nature. MacCallum et al. 1996 (J. Mol. Biol. 262:732-745) disclose the presence of planar surfaces on antibodies, but teach only modification of antibody structures not antigen structures. Further, the disclosure of MacCallum et al. is directed to antibodies and different kinds of antigens, such as carbohydrates and peptides, in general and does not teach anything particular on the binding between IgE antibodies and allergenic polypeptides or the surface structures of these polypeptides.